I. Serine Proteases
Serine proteases (E.C. 3.4.21) are the sub-sub class of endopeptidases that use serine as the nucleophile in peptide bond cleavage (Barrett, A.J., In: Proteinase Inhibitors, Ed. Barrett, A.J. et al, Elsevier, Amsterdam, pages 3-22 (1986); and Hartley, B.S., Ann. Rev. Biochem., 29:45-72 (1960)).
Serine proteases are well known in the art and two superfamilies of serine proteases, i.e., the chymotrypsin superfamily and the Streptomyces subtilisin superfamily, have been observed to date (Barrett, A.J., In: Proteinase Inhibitors, Ed. Barrett, A.J. et al, Elsevier, Amsterdam, pages 3-22 (1986); and James, M.N.G., In: Proteolysis and Physiological Regulation, Ed. Ribbons, D.W. et al, Academic Press, New York, pages 125-142 (1976)).
Examples of serine proteases of the chymotrypsin superfamily include tissue-type plasminogen activator (hereinafter "t-PA"), trypsin, trypsin-like protease, chymotrypsin, plasmin, elastase, urokinase (or urinary-type plasminogen activator (hereinafter "u-PA")), acrosin, activated protein C, Cl esterase, cathepsin G, chymase and proteases of the blood coagulation cascade including kallikrein, thrombin, and Factors VIIa, IXa, Xa, XIa and XIIa (Barrett, A.J., In: Proteinase Inhibitors, Ed. Barrett, A.J. et al, Elsevier, Amsterdam, pages 3-22 (1986); Strassburger, W. et al, FEBS Lett., 157:219-223 (1983); Dayhoff, M.O., Atlas of Protein Sequence and Structure, Vol. 5, National Biomedical Research Foundation, Silver Spring, Maryland (1972); and Rosenberg, R.D. et al, Hosp. Prac., 21:131-137 (1986)).
Some of the serine proteases of the chymotrypsin superfamily, including t-PA, plasmin, u-PA and the proteases of the blood coagulation cascade, are large molecules that contain, in addition to the serine protease catalytic domain, other structural domains responsible in part for regulation of their activity (Barrett, A.J., In: Proteinase Inhibitors, Ed. Barrett, A.J. et al, Elsevier, Amsterdam, pages 3-22 (1986); Gerard, R.D. et al, Mol. Biol. Med., 449-457 (1986); and Blasi, F. et al, In: Human Genes and Diseases, Ed. Blasi, F., John Wiley & Sons, Ltd., pages 377-414 (1986)).
The catalytic domains of all of the serine proteases of the chymotrypsin superfamily have both sequence homology and structural homology. The sequence homology includes the total conservation of:
(i) the characteristic active site residues (e.g., Ser.sub.195, His.sub.57 ; and Asp.sub.102 in the case of trypsin);
(ii) the oxyanion hole (e.g., Gly.sub.193, Asp.sub.194 in the case of trypsin); and
(iii) the cysteine residues that form disulfide bridges in the structure (Hartley, B.S., Symp. Soc. Gen. Microbiol., 24:152-182 (1974)).
The structural homology includes:
(i) the common fold that consists of two Greek key structures (Richardson, J., Adv. Prot. Chem., 34:167-339 (1981));
(ii) a common disposition of catalytic residues; and
(iii) detailed preservation of the structure within the core of the molecule (Stroud, R.M., Sci. AM., 231:24-88 (1974)).
A comparison of the sequences of the members of the chymotrypsin superfamily reveals the presence of insertions or deletions of amino acids within the catalytic domains (see for example, FIG. 1). In all cases, these insertions or deletions map to the surface of the folded molecule and thus do not effect the basic structure of the molecule (Strassburger, W. et al, FEBS Lett., 157:219-223 (1983)).
II. Serine Protease Inhibitors
Serine protease inhibitors are well known in the art and are divided into the following families: (i) the bovine pancreatic trypsin inhibitor (Kunitz) family, also known as basic protease inhibitor (Ketcham, L.K. et al, In: Atlas of Protein Sequence and Structure, Ed. Dayhoff, M.O., pages 131-143 (1978) (hereinafter "BPTI"), (ii) the Kazal family, (iii) the Streptomyces subtilisin inhibitor family (hereinafter "SSI"), (iv) the serpin family, (v) the soybean trypsin inhibitor (Kunitz) family, (vi) the potato inhibitor family, and (vii) the Bowman-Birk family (Laskowski, M. et al, Ann. Rev. Biochem., 49:593-626 (1980); Read, R.J. et al, In: Proteinase Inhibitors, Ed. Barrett, A.J. et al, Elsevier, Amsterdam, pages 301-336 (1986); and Laskowski, M. et al, Cold Spring Harbor Symp. Quant. Biol., LII:545-553 (1987)).
Crystallographic data are available for a number of intact inhibitors including members of the BPTI, Kazal, SSI, soybean trypsin and potato inhibitor families, and for a cleaved form of the serpin alpha-1-antitrypsin (Read, R.J. et al, In: Proteinase lnhibitors, Ed. Barrett, A.J. et al, Elsevier, Amsterdam, pages 301-336 (1986)). Despite the fact that these serine protease inhibitors are proteins of diverse size and sequence, the intact inhibitors studied to date all have in common a characteristic loop extending from the surface of the molecule that contains the recognition sequence for the active site of the cognate serine protease (Levin, E.G. et al, Proc. Natl. Acad. Sci. USA, 80:6804-6808 (1983)). The structural similarity of the loops in the different serine protease inhibitors is remarkable (Papamokos, E. et al, J. Mol. Biol., 158:515-537 (1982)). Outside of the active site loop, the serine protease inhibitors of different families are generally unrelated structurally, although the Kazal family and Streotomvces subtilisin family of inhibitors display some structural and sequence similarity.
Many of the serine protease inhibitors have a broad specificity and are able to inhibit both the chymotrypsin superfamily of proteases, including the blood coagulation serine proteases, and the Streptomyces subtilisin superfamily of serine proteases (Laskowski, M. et al, Ann. Rev. Biochem., 49:593-626 (1980)). The specificity of each inhibitor is thought to be determined primarily by the identity of the amino acid that is immediately amino-terminal to the site of potential cleavage of the inhibitor by the serine protease. This amino acid, known as the P site residue, is thought to form an acyl bond with the serine in the active site of the serine protease (Laskowski, M. et al, Ann. Rev. Biochem., 49:593-626 (1980)).
A. The BPTI Family
Serine protease inhibitors belonging to the BPTI family include BPTI, snake venom inhibitor, inter-alpha inhibitor, and the A4 amyloid precursor A4695 (Laskowski, M. et al, Ann. Rev. Biochem., 49:593-626 (1980); Read, R.J. et al, In: Proteinase Inhibitors, Ed. Barrett, A.J. et al, Elsevier, Amsterdam, pages 301-336 (1986); and Ponte, P. et al, Nature, 331:525-527 (1988)). Examples of serine proteases and their cognate BPTI family inhibitors are listed in Table I below.
TABLE I ______________________________________ Serine Protease Cognate BPTI Inhibitior ______________________________________ Trypsin BPTI Snake venom inhibitor Inter-alpha inhibitor (Unknown) A4 amyloid precursor A4695 protease nexin II ______________________________________
B. The Kazal Family
Serine protease inhibitors belonging to the Kazal family include pancreatic secretory inhibitor, ovomucoid and seminal plasma acrosin inhibitor (Laskowski, M. et al, Ann. Rev. Biochem., 49:593-626 (1980); Read, R.J. et al, In: Proteinase Inhibitors, Ed. Barrett, A.J. et al, Elsevier, Amsterdam, pages 301-336 (1986); and Laskowski, M. et al, Cold Spring Harbor Symp. Quant. Biol., LII: 545-553 (1987)). Examples of serine proteases and their cognate Kazal family inhibitors are listed in Table II below.
TABLE II ______________________________________ Serine Protease Cognate Kazal Inhibitor ______________________________________ Trypsin Pancreatic secretory inhibitor Ovomucoid Seminal plasma acrosin inhibitor Acrosin Ovomucoid Seminal plasma acrosin inhibitor ______________________________________
C. The Streptomyces Subtilisin Inhibitor
Serine protease inhibitors belonging to the Streptomyces subtilisin inhibitor family include inhibitors obtained from Streptomyces albogriseolus and plasminostreptin (Laskowski, M. et al, Ann. Rev. Biochem., 49:593-626 (1980)). Examples of serine proteases and their cognate Streptomyces subtilisin class inhibitors ar listed in Table III below.
TABLE III ______________________________________ Serine Protease Cognate SSI Inhibitor ______________________________________ Subtilisin BPN' Streptomyces albogriseolus inhibitor Plasmin Plasminostreptin Trypsin Plasminostreptin ______________________________________
D. The Serpin Family
Serine protease inhibitors belonging to the serpin family include the plasminogen activator inhibitors PAI-1, PAI-2 and PAI-3, Cl esterase inhibitor, alpha-2-antiplasmin, contrapsin, alpha-1-antitrypsin, antithrombin III, protease nexin I, alpha-1-antichymotrypsin, protein C inhibitor, heparin cofactor II and growth hormone regulated protein (Carrell, R.W. et al, Cold Spring Harbor Symp. Quant. Biol., 52:527-535 (1987); Sommer, J. et al, Biochem., 26:6407-6410 (1987); Suzuki, K. et al, J. Biol. Chem., 262:611-616 (1987); and Stump, D.C. et al, J. Biol. Chem., 261:12759-12766 (1986)).
The inhibition of serine proteases by serpins has been reviewed in Travis, J. et al, Ann. Rev. Biochem., 52:655-709 (1983); Carrell, R. W. et al, Trends Biochem. Sci., 10:20-24 (1985); Sprengers, E.D. et al, Blood, 69:381-387 (1987); and Proteinase Inhibitors, Ed. Barrett, A.J. et al, Elsevier, Amsterdam (1986).
Examples of serine proteases and their cognate serpin inhibitors are listed in Table IV below.
TABLE IV ______________________________________ Serine protease Cognate Serpin Inhibitor ______________________________________ Activated protein C Protein C inhibitor PAI-1 Bat PA PAI-1, PAI-2, PAI-3 C1 esterase C1 esterase inhibitor Cathepsin G Alpha-1-antitrypsin Alpha-1-antichymotrypsin Chymase Alpha-1-antichymotrypsin Chymotrypsin Alpha-1-antichymotrypsin Alpha-2-antiplasmin Contrapsin Coagulation factors Antithrombin III (VIIa, IXa, Xa, XIa, Cl esterase inhibitor XIIa) Elastase Alpha-1-antitrypsin Kallikrein C1 esterase inhibitor Alpha-1-antitrypsin Plasmin Alpha-2-antiplasmin Thrombin Antithrombin III Heparin cofactor II t-PA PAI-1, PAI-2, PAI-3 Trypsin Alpha-1-antitrypsin Growth hormone regulated protein Trypsin-like protease Protease nexin I u-PA PAI-1, PAI-2, PAI-3 ______________________________________
E. The Soybean Trypsin Inhibitor Family
A single example of the soybean trypsin inhibitor family, purified from soybeans, has been sequenced. Its complex with bovine pancreatic trypsin has been studied (Sweet, R.M. et al, Biochem., 13:4214-4228 (1974)).
F. The Potato Inhibitor Family
Serine protease inhibitors belonging to the potato inhibitor family include inhibitors from potatoes, barley and leeches (Read, R.J. et al, In: Proteinase Inhibitors, Ed. Barrett, A.J. et al, Elsevier, Amsterdam, pages 301-336 (1986)). Examples of serine proteases and their potato inhibitors are listed in Table V below.
TABLE V ______________________________________ Serine Protease Potato Inhibitor ______________________________________ Chymotrypsin Barley chymotrypsin inhibitor Subtilisin Novo Barley chymotrypsin inhibitor Subtilisin Carlsberg Leech inhibitor eglin ______________________________________
G. The Bowman-Birk Inhibitor Family
Serine protease inhibitors belonging to the Bowman-Birk inhibitor family include homologous proteins from legumes (Laskowski, M. et al, Ann. Rev. Biochem., 49:593-626 (1980)). Examples of serine proteases and their Bowman-Birk inhibitors are listed in Table VI below.
TABLE VI ______________________________________ Serine Protease Bowman-Birk Inhibitor ______________________________________ Trypsin Lima bean inhibitor IV Elastase Garden bean inhibitor Chymotrypsin Adzuki bean inhibitor II ______________________________________
III. Serine Protease-Inhibitor Complexes
Serine protease inhibitors of all families form stable 1:1 complexes with their cognate serine proteases. These complexes dissociate only slowly (hours to days) (Laskowski, M. et al, Ann. Rev. Biochem., 49:593-626 (1980); and Levin, E.G., Proc. Natl. Acad. Sci. USA, 80:6804-6808 (1983)). For all serine protease inhibitors, except the serpins, the dissociation products are a mixture of the intact and cleaved inhibitor molecules. On the other hand, since dissociation of serine protease-serpin complexes appears to yield only cleaved inhibitor molecules, serpins are thought to utilize a mechanism somewhat distinct from that of the other serine protease inhibitors.
Structural data are available for several serine protease-inhibitor complexes, including trypsin-BPTI, chymotrysin-ovomucoid inhibitor, chymotrypsin-potato inhibitor, and Streptomyces subtilisin-Streptomyces subtilisin inhibitor (Read, R.J. et al, In: Proteinase Inhibitors, Ed. Barrett, A.J. et al, Elsevier, Amsterdam, pages 301-336 (1986)). Examination of these structures reveals remarkable similarities in the specific interactions between each inhibitor and its cognate serine protease, despite the diverse sequences of the inhibitors. This structural similarity has suggested in the present invention that even when crystal structures are not available, it may be possible to predict the amino acid interactions occurring between an inhibitor and its cognate serine protease.
As discussed above, the inhibitors contain a reactive center that serves as a competitive substrate for the active site of the serine protease. Attack on the peptide bond between the P.sub.1 -P.sub.1 ' residues of the reactive center (e.g., Arg.sub.346 -Met.sub.347 in the case of PAI-1) does not lead to the normal, rapid dissociation of the products from the serine protease but, rather, to the establishment of a stable serine protease-inhibitor complex, probably by formation of a covalent bond between the serine of the active site of the protease and the P.sub.1 residue of the inhibitor (Laskowski, M. et al, Ann. Rev. Biochem., 49:593-626 (1980)). This mechanism indicates that the reactive center of an inhibitor, such as PAI-1, must fit tightly and precisely into the active site of the serine protease. However, to date, there are no X-ray crystallographic data on PAI-1, its cognate serine protease, t-PA, or the t-PA/PAI-1 complex. Thus, the exact nature of the interactions between this pair of proteins is unknown. There is a similar lack of structural information about other serpins or serpin-serine protease complexes.
IV. Utility of Serine Proteases
A particularly important serine protease of the chymotrypsin superfamily is t-PA. Most members of the chymotrypsin family of serine proteases are synthesized as inactive, single chain precursors or zymogens. Subsequent cleavage of a specific peptide bond converts these precursors into fully active two-chain enzymes. By contrast, the single-chain form of t-PA displays significant catalytic activity and its V.sub.max for generation of plasmin from plasminogen is only about 3-5 fold lower than that of two-chain t-PA (Boose, J. A. et al, Biochem., 28:635-643 (1988); and Petersen, L.C. et al, Biochim. Biophys. Acta, 952:245-254 (1988)).
t-PA is currently being used, via intracoronary or intravenous administration, to treat myocardial infarction, pulmonary embolism, and deep venous thrombosis, although it does not work directly to dissolve thrombi (blood clots). Rather, t-PA promotes cleavage of the peptide bond between Arg.sub.560 and Val.sub.561 of plasminogen (Robbins, K.C. et al, J. Biol. Chem., 242:2333-2342 (1967)), thereby converting the inactive zymogen into the powerful but non-specific protease, plasmin, which then degrades the fibrin mesh work of the blood clot (Bachmann, F. et al, Semin. Throm. Haemost., 43:77-89 (1984); Gerard, R.D. et al, Mol. Biol. Med., 3:449-557 (1986); and Verstraete, M. et al, Blood, 67:1529-1541 (1986)).
t-PA produces local fibrinolysis without necessarily depleting systemic fibrinogen. This is because t-PA has the ability to bind directly to fibrin, forming a fibrin-t-PA complex whose affinity for plasminogen is increased approximately 500 fold (Ranby, M. et al, Biochim. Biophys. Acta, 704:461-469 (1982); and Rijken, D.C. et al, J. Biol. Chem., 257:2920-2925 (1982)). Thus, binding of intravenously-administered t-PA to coronary thrombi, where plasminogen is also present in high concentration (Wiman, B. et al, Nature, 272:549-550 (1978)), results in efficient production of plasmin at the site of the thrombus where it will do the most good.
At present, t-PA is administered in the form of an initial bolus that is followed by sustained infusion. The total amount of enzyme administered during a standard 3 hour treatment is generally about 50-100 mg. Such large amounts are apparently required for two reasons: first, to counterbalance the effects of rapid clearance of t-PA from the circulation by hepatic cells (Krause, J., Fibrinolysis, 2:133-142 (1988)), and second, to overcome the effects of comparatively high concentrations of serine protease inhibitors that are present in plasma and platelets (Carrell, R.W. et al, In: Proteinase Inhibitors, Ed. Barrett, A.J. et al, Elsevier, Amsterdam, pages 403-420 (1986)).
The major physiological inhibitor of t-PA is the serpin, PAI-1, a glycoprotein of approximately 50 kd (Pannekoek, H. et al, EMBO J., 5:2539-2544 (1986); Ginsberg, D. et al, J. Clin. Invest., 78:1673-1680 (1980); and Carrell, R. W. et al, In: Proteinase Inhibitors, Ed. Barrett, A. J. et al, Elsevier, Amsterdam, pages 403-420 (1986)). PAI-1 has been implicated as the cause of reduced fibrinolytic capacity of plasma from survivors of myocardial infarctions (Hamsten, A. et al, New Eng. Med., 313:1557-1563 (1985)). In addition, PAI-1 is an acute phase reactant and the elevated levels associated with myocardial infarction may attenuate the fibrinolytic activity of substantial amounts of the t-PA remaining in the plasma after therapeutic infusion of the t-PA (Lucore, C.L. et al, Circ., 77:660-669 (1988)). The second-order rate constant for association of PAI-1 with t-PA is extremely high (Hekman, C. et al, Arch. Biochem. Biophys., 262:199-210 (1988)) and accounts for the initial, " fast-phase" inhibition of t-PA by human plasma (Colucci, M. et al, J. Lab. Clin. Med., 108:53-59 (1986)). The rapid neutralization of t-PA by PAI-1 in vivo. may therefore contribute to coronary restenosis after thrombolytic therapy, a complication that affects between 10% and 35% of patients treated for acute myocardial infarction (Chesebro, J.H. et al, Circ , 76:142-154 (1987)).
Although the association constants of other serpins, such as Cl esterase inhibitor and alpha-2-antiplasmin, with t-PA are orders of magnitude lower than that of PAI-1 (Ranby, M. et al, Throm. Res., 27:175-183 (1982); and Hekman, C. et al, Arch. Biochem. Biophys., 262:199-210 (1988)), these serpins nevertheless bind to infused t-PA (Lucore, C.L. et al, Circ., 77:660-669 (1988)) and may attenuate the beneficial pharmacological properties of t-PA.
Besides t-PA and PAI-1, many other serine protease-serpin pairs are of great medical importance. For example u-PA, like t-PA, is useful in the treatment of myocardial infarction and is subject to inhibition by the same serine protease inhibitors as t-PA.
Thrombin, the serine protease used topically to promote blood clotting of wounds, is a procoagulant. Its cognate serpin, antithrombin III, is an anti-coagulant that specifically inhibits a number of serine proteases that participate in the blood coagulation cascade, including thrombin and Factors IXa, Xa, XIa and XIIa (Heimburger, N. et al, In: Proceedings of the International Research Conference on Proteinase Inhibitors, Ed. Fritz, H. et al, Walter de Gruyter, New York, pages 1-22 (1971); Kurachi, K. et al, Biochem., 15:373-377 (1976); Kurachi, K. et al, Biochem., 16:5831-5839 (1977); and Osterud, B. et al, Semin. Thromb. Haemost., 35:295-305 (1976)). Antithrombin III has been used therapeutically to treat disseminated intravascular coagulation. The activation of protein C by thrombin results in the self-limitation of the blood coagulation process because activated protein C inactivates coagulation factors Va and VIIIa, and is itself inhibited by its cognate serpin, protein C inhibitor.
Kallikrein, which functions to induce uterine contraction, to increase vascular permeability, and to initiate the intrinsic pathway of blood coagulation, is subject to inhibition by the serpin alpha-1-antitrypsin, one of the more important serpins.
Alpha-1-antitrypsin also inhibits leukocyte elastase and cathepsin, as well as trypsin, chymotrypsin and plasin (Heimburger, N. et al, In: Proceedings of the International Research Conference on Proteinase Inhibitors, Ed. Fritz, H. et al, Walter de Gruyter, New York, pages 1-47 (1971); Janoff, A., Am. Rev. Resp. Dis., 105:121-127 (1972); and Ohlsson, K. et al, Eur. J. Biochem., 36:473-481 (1973)). The genetic deficiency of alpha-1-antitrypsin is directly related to emphysema (Carrell, R.W. et al, Trends Biochem. Sci., 10:20-24 (1985)) and alpha-1-antitrypsin replacement therapy has been used to treat emphysema (Marx, J.L., Science, 243:315-316 (1989)).